The purpose of the experiments proposed in this application is to continue to explore the intracellular relationships between energy supply and ion transport in three interrelated areas: 1) the metabolic effects of diuretics, 2) the effects of sodium on kidney mitochondria, and 3) the mechanism of energy coupling in mitochondria. The present application proposes an investigation of the effects of ethacrynic acid and furosemide on kidney metabolism by performing in vivo measurements of renal metabolite levels following diuretic administration. The renal artery will be clamped and subsequently released to alter levels of ATP and intermediates of the oxidative and glycolysis pathways. Slices of canine renal cortex and medulla will be rapidly freeze-clamped. The concentrations of metabolic intermediates of the glycolytic and oxidative pathways, adenine nucleotides, and the redox state of components of the electron transport chain will be determined. To determine the site of diuretic action, the effects of ouabain and cyanide will be compared with the diuretics. The levels of the diuretics in renal tissue will also be measured. Since most of the energy for sodium transport is generated by oxidative metabolism, in vitro studies of the effects of ethacrynic acid and furosemide on respiration of isolated renal tubules will be continued. Furthermore, the mechanism of diuretic-induced inhibition of Site II of the mitochondrial electron transport chain will be investigated, as well as effects on the mitochondrial adenine nucleotide translocase. A study of the effects of sodium on isolated kidney mitochondria will be continued with emphasis on the mechanism of sodium- stimulated ATPase activity and energy-linked sodium extrusion. Finally, previous investigations concerning the mechanism of action of uncoupling agents and the relationship of the electron transport chain to the site of ATP synthesis in mitochondria will be continued.